60
Circular Dichroism in Protein Analysis
C
C
a
N
C
a
H
O
m
NV1
(
55
°
)
m
NV2
(60
°
)
m
n
p
*
Fig. 3
The n
π
and
ππ
transitions of the peptide
chromophore in the far UV
region.
µ
is the electrical
transition dipole moment.
m
is
the magnetic transition
dipole moment.
aprotein
,then
π
transition is not strictly
forbidden, but it is very weak. In solvents
that lack hydrogen-bond donors, the n
π
transition appears near 230 nm, while in
aqueous solution, it is around 210 nm.
The n
0
π
transition occurs at
140 nm
and is polarized perpendicular to the
carbonyl bond.
The
ππ
transitions at 190 and 140 nm
are electrically allowed. The Frst
ππ
tran-
sition, the NV
1
band, involves excitation
of an electron from the nonbonding
π
orbital,
π
0
,t
ot
h
e
π
orbital. In sec-
ondary amides, the NV
1
band is observed
around 185 to 190 nm, whereas in ter-
tiary amides, it is closer to 200 nm. The
second
ππ
transition, the NV
2
band, at
140 nm involves electronic excitation from
the bonding
π
orbital,
π
+
,tothe
π
or-
bital. The transition dipole moments of the
two
ππ
transitions in secondary amides
are approximately parallel (NV
1
)andper
-
pendicular (NV
2
) to the C–N bond. The
electric transition dipole moment of the
π
0
π
transition (NV
1
)isabou
t3
.1Dand
that of the
π
+
π
transition (NV
2
)isabout
1.8 D. In an
α
-helix, the electric dipole
coupling of the
π
0
π
(NV
1
) transitions on
neighboring residues results in two bands
i
nt
h
eCDs
p
e
c
t
r
um
,o
n
ea
t
190 nm
and the other, a long-wavelength com-
ponent, at
208 nm. Interactions among
the amide n
π
and
ππ
transitions in a
peptide are relatively delocalized and are
signiFcantly affected by environmental or
local perturbations.
The arrangement of peptide bonds in a
protein largely determines its CD spec-
trum in the far UV. The main-chain
dihedral angles deFne the relative orien-
tation of the backbone chromophores. If
the dihedral angles for each amino acid
residue were the same, the protein would
form a perfectly regular helix (a
β
-strand
may be viewed as a very narrow helix).
Alternatively, an arbitrary set of (sterically
allowed) dihedral angles would generate a
random coil conformation. In reality, most
secondary structures lie between these two
extremes. The origins of the electronic CD
of proteins can be understood by dividing
the biopolymer into subunits, which do
not have signiFcant electronic exchange
among them. This basic concept under-
lies theoretical calculations of protein CD
spectra. Protein CD spectra arise from
three types of interactions between
ππ
and n
π
transitions of amides. Exciton
interactions occur between degenerate or
nearly degenerate
ππ
transitions on dif-
ferent peptide groups. An n
π
transition
and a
ππ
transition on different peptide
groups may mix; this is also referred to as
µ
mcoup
l
ing
. ±inally, the n
π
and
ππ
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